Resistance of differentiated human airway epithelium to infection by rhinovirus

Abstract

in vitro studies of the effects of rhinovirus on human airway epithelium have used cells grown under conditions known to produce low levels of differentiation. The relevance of the results to native epithelium is questionable. Here we grew primary cultures of human tracheal or nasal epithelium under three conditions. One condition produced pseudostratified, mucociliary cells virtually indistinguishable from native epithelium. The other two conditions produced undifferentiated squamous cells lacking cilia. Cells were infected for 6 h with rhinovirus-16. After a 24-h incubation period, we determined levels of viral RNA in the cells, numbers of infectious viral particles released in the mucosal medium, expression of a variety of epithelial cytokines and other proteins, release of IL-6 and IL-8, and transepithelial electrical resistance and voltage. After infection, levels of viral RNA in the poorly differentiated cells were 30 or 130 times those in the differentiated. Furthermore, expression of mRNA for inflammatory cytokines, release of infectious particles, and release of IL-6 and IL-8 were closely correlated with the degree of viral infection. Thus well-differentiated cells are much more resistant to viral infection and its functional consequences than are poorly differentiated cells from the same source. ion transport; porous-bottomed inserts; short-circuit current; transepithelial electrical resistance RHINOVIRUS (RV) causes severalfold increases in the output of a variety inflammatory cytokines from human airway epithelial cell lines (29,41) and from primary cultures of human airway epithelium (18,23,31,39). However, the cell lines used (A549 and BEAS-2B) generally do not form tight junctions (17,25), and the primary cultures were grown on solid supports, an approach known to produce highly undifferentiated cells (36). We questioned whether results obtained on such cultures were really representative of viral infection of native epithelium. Accordingly, we have grown cells from the same tracheas and nasal scrapings under several different culture conditions to achieve a range of differentiation from a squamous to a full-blown pseudostratified, mucociliary phenotype. We then compared the susceptibility of these various cultures to viral infection. METHODS

Interleukin-13–Induced Mucous Metaplasia Increases Susceptibility of Human Airway Epithelium to Rhinovirus Infection

“…By contrast, in mucociliary cultures, 1.6% 6 0.09% of cells were infected, and numbers of foci of infection were almost always one per cell, and never more than three ( Figure 4C). These results are consistent with our previous studies indicating that squamous cultures of human airway epithelium show levels of viral infection (measured from infectious particles released and levels of viral RNA in cell lysates) z100-fold greater than those of mucociliary cultures derived from the same tracheas (9). After treatment of mucociliary cultures with IL-13, numbers of rhinovirus-infected cells increased by z2.5-fold ( Figure 4D).…”

“…This hypothesis already receives considerable support from our earlier finding of greatly increased levels of infection in squamous cultures (9). However, in cultures grown under conditions that produce mucociliary differentiation, it is well-established that the complexity of the apical membrane increases progressively over the first 28 days in culture (10, 13).…”

“…We previously showed that the degree of viral infection depends on the type of differentiation of human airway epithelial cell cultures. Specifically, after exposure to the same multiplicity of infection (MOI) of rhinovirus, squamouscell cultures produced z100 times as much virus as cultures of healthy mucociliary phenotype (9). We therefore hypothesized that the mucous metaplasia seen in asthma also makes the epithelium more susceptible to viral infection.…”

Cigarette Smoke Decreases Innate Responses of Epithelial Cells to Rhinovirus Infection

“…Well-differentiated airway epithelial cells were shown to be more resistant to HRV infection than the same cells in undifferentiated monolayers (45). However, a recent study reported that various types of differentiated bronchial epithelial cells differ in their susceptibility to RV infection (46).…”

All-trans retinoic acid mediates DUOX2 expression and function in respiratory tract epithelium

“…For RV1B infection, a multiplicity of infection (MOI) of 1 was used by adding a volume of RV1B that provided 1 ϫ 10 6 plaqueforming units (PFU) to 1 ϫ 10 6 respiratory tract epithelial cells. This protocol was based on previously published techniques (20).…”